CN103893758A - Composition comprising chitosan for ocular administration of vaccine(s) to avians - Google Patents
Composition comprising chitosan for ocular administration of vaccine(s) to avians Download PDFInfo
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Abstract
The invention relates to vaccine compositions comprising chitosan and a vaccine which are administered to avian species via the ocular route, and methods or programs of vaccination including use of chitosan in order to increase or enhance cell-mediated immunity in avian species and persistence of the vaccine avian tissues.
Description
The application is to be the divisional application of JIUYUE in 2009 4 days, the application number application of the same name that is 200980138635.5 the applying date.
Invention field
The present invention relates to comprise chitosan (chitosan) and the vaccine combination of vaccine and the method for inoculation or program (it comprise use chitosan to increase or to strengthen the immunne response to vaccine in avian species) by what use to avian species through eye approach.
Background of invention
Vaccine is to use for example, preparation the active immunity of specified microorganisms (antibacterial or virus or parasite) is strengthened by induction the antigenicity material of the resistance to infecting to experimenter.Vaccine and adjuvant can be used to strengthen immunne response altogether.The example of adjuvant comprises the chemistry of replying and the polypeptide immune stimulant of enhancing immune system to antigen.This type of adjuvant can comprise such as aluminium hydroxide, aluminum phosphate, plant and animal oil and mineral oil, bacteriotoxin, chitin (chitin) derivant and chitosan etc., adjuvant can be used together with vaccine with the amount that is enough to strengthen immunne response.
Conventionally inject for example subcutaneous or intramuscular injection by parenteral and use vaccine and adjuvant.Especially, for the infection for the treatment of upper respiratory tract, also can proceed to directly the using of nasal mucosa of many animals species.For example, European patent EP 865297B1 has described the protein from pathogen or polysaccharide antigen and has been applied to altogether mice as the chitosan intranasal of adjuvant.By aerosol, drop or insufflation, this type of intranasal compositions is formulated as to the dry powder of microsphere form, and allows to strengthen IgA mucosa body fluid and IgG systemic immune response by other continuous intranasal administration several times.
Applicant has now found that, the chitosan being used in combination with bird vaccine in identical vaccine program has and greatly strengthens cell-mediated immunne response and maintain the high body fluid of vaccinated birds and the potentiality of systemic immune response (when by the time that eye approach is used) simultaneously.In addition, find, chitosan acts on birds conjunctiva and causes vaccine retaining in birds tissue (persistence) significantly to increase.
Summary of the invention
The present invention relates to be administered to through eye the vaccine combination of avian species, the chitosan that it comprises one or more vaccines and effective adjuvant amount.The invention still further relates to the method for immune avian species, it comprises to avian species uses vaccine combination through eye, and being combined in of one or more vaccines and chitosan manufactured for use the purposes so that immune avian species is resisted the vaccine combination of infectious disease effectively through eye.The invention still further relates to birds vaccinating agents box, it comprises vaccine combination and the instrument of using through eye or device for described vaccine combination.
Summary of drawings
Fig. 1: the timeline that illustrates inoculation.
Fig. 2: illustrate antigen and recall (antigen recall) scheme.
Fig. 3: show as thering is or do not have chitosan using through eye
the yield level (optical density) of the chicken interferon-gamma of measuring for 2 to 5 weeks after VITAPEST L (Ceva Phylaxia, Hungary).
Fig. 4: show by accepting to have or do not have chitosan through eye approach
uNI L or
in the birds tissue of the SPF chicken of VITAPEST L with egg-infective dose (EID
50) titre of newcastle (ND) vaccine strain that represents.
Fig. 5: show by accepting to have or do not have chitosan through eye approach
uNI L (Ceva Phylaxia, Hungary) or
in the birds tissue of the conventional laying hen of VITAPEST L with egg-infective dose (EID
50) titre of newcastle disease vaccine strain that represents.
Fig. 6 A and 6B: be presented at the immunity with the immunocompetence after newcastle disease vaccine alive (NDV) inoculation SPF chicken (Fig. 6 A) and conventional laying hen (Fig. 6 B) and specific cell mediation.By through eye approach, in the time of an age in days with independent
vITAPEST L (black post) or with chitosan combination
vITAPEST L (Lycoperdon polymorphum Vitt post) inoculates birds.Nonvaccinated animal indicates with white post.With PMA/Iono mitogen (0.1 μ g/ml) gp-NDV recall antigen (1 μ g/ml) stimulation splenocyte, gather in the crops afterwards the supernatant of the cell being stimulated the activation of 72 hours.Catch the output of ELISA mensuration ChIFN γ by ChIFN γ.Result is corresponding to the mean+SD (n=5) of the optical density on each time point (O.D.).There are different upper target mean+SD significant differences (P<0.05).
Fig. 7 A and 7B: be presented at the antibody-mediated immunity of tear (lachrymal) after live ND vaccination SPF chicken (Fig. 7 A) and conventional laying hen (Fig. 7 B).In the time of an age in days, pass through (the Lycoperdon polymorphum Vitt post) that combine through independent (the black post) of eye approach or with chitosan
vITAPEST L inoculates birds.Nonvaccinated animal indicates with white post.Data are illustrated in the mean+SD (n=5) of the upper absorbance (O.D.) of measuring by ELISA of time of specifying after inoculation.Tear sample is diluted with 1:4 (for IgA/G) and 1:32 (for IgM).There is the mean+SD significant difference (P<0.05) on different upper target time points.N.D.=is undeterminate due to the limited amount sample of residue after NDV specificity IgA and IgG measurement.
Fig. 8 A and 8B: be presented at the antibody-mediated immunity of bile after live ND vaccination SPF chicken (Fig. 8 A) and conventional laying hen (Fig. 8 B).In the time of an age in days, by through eye approach, with independent (black post) or with (the Lycoperdon polymorphum Vitt post) of chitosan combination
vITAPEST L inoculates birds.Nonvaccinated animal indicates with white post.Data are illustrated in the mean+SD (n=5) of the upper absorbance (O.D.) of measuring by ELISA of time of specifying after inoculation.Bile sample is diluted with 1:50.There is the mean+SD significant difference (P<0.05) on different upper target time points.
Fig. 9 A and 9B: be presented at ND vaccination SPF chicken (Fig. 9 A) and the antibody-mediated immunity of conventional laying hen (Fig. 9 B) metaduodenum of living.In the time of an age in days, (the Lycoperdon polymorphum Vitt post) combining with independent (black post) or with chitosan
vITAPEST L inoculates birds.Nonvaccinated animal indicates with white post.Data are illustrated in the mean+SD (n=5) of the upper absorbance (O.D.) of measuring by ELISA of time of specifying after inoculation.In the undiluted supernatant of in vitro Duodenal Tissues culture, measuring Ig replys.There is the mean+SD significant difference (P<0.05) on different upper target time points.
Detailed Description Of The Invention
The invention provides the vaccine combination for using through eye to avian species, the chitosan that said composition comprises one or more vaccines and effective adjuvant amount.As mentioned above, have surprisingly been found that, after eye is used altogether, chitosan and vaccine have greatly strengthened the cell-mediated immunne response of avian species and have caused longer retain of vaccine in the particular organization of vaccinated birds.Thereby vaccine combination of the present invention is effective especially for the Long-Term Protection that improves the anti-poultry disease of avian species.
Those skilled in the art understand term " effectively adjuvant amount " completely, it comprises the amount of the chitosan of the immunne response that can stimulate the anti-antigen of using through eye,, increase the amount of the immunne response (for example, as according to the measurement that retains of vaccine in the level of the gamma interferon being produced by splenocyte and birds tissue) of the vaccine combination used through eye.In the situation that chitosan exists, observe the gamma interferon level of generation and vaccine and organize the remarkable increase of retention time.For example, the increase of cell response also can be passed through cell proliferation test (by recalling after stimulation at antigen
3h thymidine is to the measurement of mixing in somatoblast), by measuring cellular cytoxicity activity (mensuration of radioisotopic release within a period of time) to radiolabeled target cell of CTL cell or testing to prove by macrophage migration.
According to the present invention, vaccine can be any birds pathogenicity or non-pathogenic microorganisms, for example virus, antibacterial, any other parasite or antigen.This type of vaccine can be the attenuated microorganisms of living or the chimeric or recombinant microorganism of killed deactivation microorganism (complete microorganism or the subunit of microorganism), deactivation, destroyed microorganism, the microorganism of sudden change, defective microorganism or its combination.Vaccine can also be to comprise for example antigen of virus, antibacterial or parasitic epi-position or antigenic portions of microorganism structure, for example, from antigenic protein, the recombiant protein of pathogen, for example preparation of polysaccharide, lipopolysaccharide and glycoprotein of part of preferred virus antigen for example viral capsid proteins, cell wall protein, peptide or antibacterial or parasite structure.Vaccine can also be DNA or recombinant DNA, and described DNA produces the antigen from pathogen after the introducing of DNA in cell.Can provide antigen with purification or unpurified form.
When vaccine is attenuation (replicability) microorganism of living for example when virus, antibacterial or other avian pathogens, the pathogen of attenuation keeps non-pathogenic matter, and can copy.Attenuation can be from natural or artificial method of attenuating.Artificial attenuation conventionally uses various technology to be included in animal alive or multiple natural medium (comprise organ, cell, containing the ovum of embryo etc.) and goes down to posterity to obtain.Artificial attenuation also can be by the ageing of dry, the culture of the organ that infects, adaptation, the genetic defect etc. of low temperature or Incubation Condition are obtained.
Vaccine can also be the microorganism of killed deactivation.Conventionally obtain the preparation of the inactivation of viruses for inoculating by chemistry or physical method.Can realize chemical ablation by processing virus with for example enzyme, formaldehyde, beta-propiolactone, Ethylenimine or derivatives thereof.Then can neutralize or the stable inactivation of viruses obtaining.Can carry out physical deactivation by virus being experienced to high-energy radiation for example UV light, X ray or gamma-rays.
Also can buy from commercial source microorganism for example virus, antibacterial or other birds parasites of this type of attenuation or deactivation.
Vaccine can be homology (for example protecting the fowl disease poison of chicken) or allos (for example protecting the turkey virus of chicken) type.Selectively, bacterin preparation can comprise the combination (being called immune combination vaccine) of active antigen (live antigen) and specific antibody.
According to the present invention, vaccine or antigen can derive from and cause by G.D.Butcher, J.P.Jacob and F.B.Mather (PS47, Veterinary Medicine-Large Animal Clinical Sciences Department, Florida Cooperative Extension Service, Institute of Food and Agricultural Sciences, University of Florida, May1999) common poultry disease (for example fowl pox (Avian Pox) of describing, newcastle, infectious bronchitis, qb, lymphatic system leucocyte hyperplasia, Marek (Marek's Disease), infectious bursal disease, infectious laryngotracheitis, egg drop syndrome, Reovirosis, infectious tenosynovitis, birds encephalomyelitis, a swollen syndrome, turkey rhinotracheitis or bird flu) virus, derive from and cause mycoplasma (mycoplasmosis), hemorrhagic septicemia, salmonellosis, the antibacterial of Bordetellosis etc. and/or derive from and cause coccidiosis, other birds parasites of campylobacteriosis.Preferred vaccine for vaccine combination of the present invention comprises complete attenuated live Strain.
Preferably, vaccine derives from Avian pneumo-encephalitis virus (NDV) or derives from the virus that causes Marek, i.e. marek's disease virus (MDV) or herpes turkey virus (HVT).Similarly, also can use recombinant hvt (rHVT) and recombinant mdv (rMDV).Can by by least one heterologous nucleic acid sequence (, corresponding to the natural HVT of being present in or MDV in the not same gene of the nucleotide sequence of gene or the DNA of its part) be integrated into viral genome this type of recombinant virus carried out to genetic modification.Selectively, can by by homologous nucleotide sequence (, corresponding to the natural HVT of being present in or MDV in the same gene of gene or the DNA of its part) be integrated into viral genome rHVT or rMDV carried out to genetic modification.
Chitosan is chitinous derivant and the linear polysaccharide that forms corresponding to GLUCOSAMINE (deacetylated unit) and the N-acetyl group-GLUCOSAMINE (acetylation unit) of the β by random distribution-(1-4)-connect.Commercially produce chitosan by the deacetylated effect of chitin (structural detail in its ectoskeleton that is Crustacean).Deacetylated degree can be measured with NMR spectrum analysis.For example, the degree of deacetylation of business chitosan is in the scope of 60-100%.
Can be by the deacetylated degree to being greater than 40% for the chitosan of vaccine combination of the present invention, preferably 50% to 90%, more preferably 70% to 95% deacetylatedly produce from chitin.This type of deacetylated chitosan can be in trade name " Sea Cure
+" under chitosan glutamate salt from Protan Biopolymer A/S, Drammen, Norway is commercially available.The molecular weight of chitosan can be between 10kD to 500kD, preferably between 50kD to 300kD and more preferably between 100kD to 300kD.
According to the present invention, chitosan can be used as effective adjuvant with the form of single carboxymethyl chitosan (Mono Carboxy Methylated Chitosan, MMC) (also referred to as carboxymethyl chitosan (CMC)) or with the form of chitosan hydrochlorate (HCl chitosan).That HCl chitosan is known in the art and for example sold by Kraeber Gmbh & Co..
For example, can be in 0.1% to 5% scope for the concentration of the HCl chitosan of vaccine combination, preferably approximately 0.5% or approximately 1%.
Vaccine combination according to the present invention can be used for effective immunity of the avian species of anti-infection property poultry disease.In fact, as mentioned above, found after eye is used, this type of vaccine combination greatly strengthens cell-mediated immunne response and vaccine the retaining in many birds tissues of birds.In fact, after inoculation one week, comprise in conjunctiva, lung, spleen, trachea, digestive tract for example duodenum, pancreas and caecal tonsils (caecal tonsil) and observe vaccine retaining in birds tissue at birds tissue.In addition,, after inoculation, particularly, in conjunctiva, lung, duodenum, pancreas and caecal tonsils, such retaining maintains 5 weeks.
The avian species that available vaccine combination of the present invention is used through eye comprise for example chicken, turkey, goose, duck, pheasant, Carnis Coturnicis japonicae or pigeon and more commonly raise or poultry (poultry) or the bird (fowl) of raising and train.
According to other aspect of the present invention, the vaccine combination that definition is herein provided is inoculation birds with the purposes in the para-infectious method of anti-fowl, and described method comprises to birds experimenter to be used and comprise vaccine and the effective vaccine combination of the chitosan of adjuvant amount through eye.
According to the present invention, can use dividually or simultaneously by eye drop, spray or aerosol chitosan and the vaccine of effective dose.Selectively, can one after the other use chitosan and vaccine to avian species, can before vaccine or after vaccine, use chitosan.
The invention still further relates to the chitosan of effective dose and the vaccine purposes for the preparation of vaccine combination, described vaccine combination is replied for increasing the bird immunity of anti-avian pathogens, wherein use chitosan and vaccine by eye drop, spray or aerosol, and wherein birds cell-mediated reply with birds tissue in pathogen retain increase.As mentioned above, vaccine can be birds parasite, virus or antibacterial.More clearly, vaccine can be attenuated microorganisms, the killed deactivation microorganism of living, or complete attenuation or deactivation microorganism, its subunit or its combination.Similarly, according to the present invention, also can separate or use chitosan and vaccine to avian species simultaneously.In the time one after the other using chitosan and pathogen, can before vaccine or after vaccine, use chitosan.
In addition; according to additional aspects of the present invention, provide by avian species through eye be applied in above definition comprise vaccine and effectively the vaccine combination of the chitosan of adjuvant amount by the vaccine combination of definition herein for strengthening the cell-mediated immunne response of protectiveness and the vaccine purposes in the method retaining of birds tissue.
The invention still further relates to the method for immune avian species, it comprises the step that vaccine combination as above is applied to the ophthalmic of avian species.The invention still further relates to by the combined administration of the chitosan of one or more vaccines and effective dose to the ophthalmic of avian species being increased to immunity cell-mediated in avian species and the method that retain of vaccine in birds tissue.Can separate, in succession or simultaneously use vaccine and chitosan.
In addition, the method according to this invention, can be undertaken using through eye by spraying, aerosol or eye drop.Preferably, after reducing gradually, maternal antibody carries out such using through eye.
Most preferably, the reinforcement of further inoculating is with the immunne response of stimulation birds, and described birds has experienced inoculation or experience inoculation in the time of age a couple of days in ovum, thereby induces the stimulation of higher immunne response.Can in succession or simultaneously carry out such inoculation reinforcement to 1 age in days or the birds in 4 week age with immunity for the first time.
Eye according to the present invention can be formulated as to liquid or dry powder with vaccine combination, to use as aerosol, spray or eye drop.Therefore, can to the head of bird, use vaccine combination to birds by Direct spraying.Selectively, as for people, one or many vaccine combinations directly can be placed in to the ophthalmic of each individual bird.Preferred composition according to the present invention is formulated as to the eye drop of liquid form.
The compositions of using as aerosol, eye drop or spray can comprise the excipient in such composition that is generally comprised within of one or more types, such as antiseptic, viscosity modifier, tension regulator, tear blocker, buffer agent, stabilizing agent, carrier, adjuvant etc., to prepare the preparation of using through eye.In aqueous medium, keep solvable in order to ensure chitosan, and guarantee that antigen is not adversely affected by the pH of too acid, preferably has the pH of scope in 5.5 to 6.5 for the solution of using through eye.Carrier can be to well known to a person skilled in the art any of many aqueous buffers, for example phosphate buffer.Can use other adjuvant.That this type of adjuvant is known in the art and can comprise oil emulsion, for example aluminium hydroxide of aluminum salt or gel or aluminum phosphate, saponin, vitamin, from the extract of bacteria wall, based on such as carbopol of polyacrylic polymer, nonionic block polymer, for example avridine of fatty acid amine (avridin) and DDA, for example dextran sulfate of polymer and deae dextran based on glucosan (dextran), biodegradable microcapsule, liposome, such as MDP of virus immunity stimulus object, LPS and glucan (glucans).
The present invention also provides product or the birds vaccinating agents box of the instrument that comprises the vaccine combination of the eye for distributing the chitosan that comprises vaccine and effective dose.More clearly, birds vaccinating agents box comprise be suitable for by vaccine combination be directly dispensed to birds eye in distributor.This type of distributor can for example be taked the form of aerosol, spray or eye drop delivery system, and can arrange only to distribute single dosage or multiple dosage to the eye of birds.Birds vaccinating agents box can comprise and comprises separately the chitosan of effective dose and the container separating of vaccine.Birds vaccinating agents box according to the present invention also can comprise having using and the technical instruction of the information of dosage about pharmaceutical composition.
Can and increase for example amount that retain of vaccine virus strain in avian species of vaccine with the immunne response of irritation cell mediation effectively and use vaccine.For example, can use vaccine to birds by one or more dosage, each dosage for example comprises 10
5to 10
8eID
50.
The existence in the birds of new hatching of more antiviral protective effects of providing and being conventionally restricted due to the transmission by maternal antibody (MDA) (shift by yolk, inhaled again and be present in little chicken serum subsequently) of existing live vaccine.During can passing through, this type of MDA disturbs inoculation with live vaccine.Therefore, the immunne response of inoculation can be delayed and/or limit in conventional chicken, as observed in systemic levels before.
Thereby, preferably after MDA weakens, use according to vaccine of the present invention, thereby allow to induce good immunne response before birds may be exposed to the strain of NDV.Most preferably, according to weakening of MDA, in the time of 2 or 3 week age, carried out booster shot.
As showed in greater detail in the following embodiments, SPF chicken (without clear and definite pathogen) or conventional laying hen to 4 groups of 1 day ages carry out inoculation experiments (Fig. 1).Use eye drop by using vaccine combination through eye approach to the chicken of all groups.The group that is called " negative chicken " is not accepted any vaccine.With a dosage
uNI L or with a dosage
vITAPEST L (both corresponding to live in attenuation Avian pneumo-encephalitis virus) inoculation second and the 3rd group.Chitosan is also used as adjuvant.Only negative and
vITAPEST L uses or does not use adjuvant to inoculate, to confirm chitosan surprising effect retaining in birds tissue to cell-mediated immunity and vaccine virus.The from the 1st to the 5th week each week collected sample.Local (BALT) immunity of assessment and confirmation.
Especially, the applicant proves, and antigen recalls that to activate be effective especially.In fact, inoculate the yield level that rear (p.v.) collects the splenocyte of vaccinated chicken for 2 to 5 weeks and it contacted to assess gamma interferon (ChIFN γ) with Newcastle Disease toxalbumin.Use ELISA test kit to carry out the measurement of chicken interferon-gamma by optical density (O.D.).The yield level of ChIFN γ represents the irritation level of splenocyte in vaccinated birds, thereby represents cell-mediated immune level (Fig. 2).
In addition, in embodiment 3 below, proved that birds according to the present invention causes through eye inoculation high to cell-mediated immune effect surprisingly.Unrestricted, for example prove the immune positive effect in the specific cell mediation of the antagonism of chitosan after eye is used Avian pneumo-encephalitis virus.Show
vITAPEST L group and use chitosan
the yield result (Fig. 3) of ChIFN γ in VITAPEST L group.Be presented at after SPF chicken inoculation second to the 5th week, used chitosan
vITAPEST L group has ratio
the cell-mediated immune level that VITAPEST L group is higher.This difference was significant at the 2nd week statistically.
In addition, proved using and induced vaccine virus strain longer retaining (Fig. 4) of (in SPF chicken and conventional laying hen) in chicken tissue through eye of vaccine taking chitosan as adjuvant.As shown in embodiment 2 below, by the Different Organs (being conjunctiva, lung, trachea, duodenum, pancreas, caecal tonsils and spleen) of the 1st week and collection in the 5th week after eye inoculation chicken is carried out to PCR in real time experiment, assess the existence of Avian pneumo-encephalitis virus after eye is inoculated SPF chicken.
First, in this type of Different Organs of SPF chicken, assessed vaccine strain virus replication.After the inoculation of SPF chicken first week, in conjunctiva, lung and spleen, observe par
uNI L vaccine virus and
the virus replication of VITAPEST L vaccine virus.
uNI L vaccine virus copies substantially in trachea, with
vITAPEST L has statistically significant difference.
vITAPEST L vaccine virus for example copies in duodenum, pancreas and caecal tonsils at digestive tract substantially, with
uNI L has statistically significant difference.At after eye inoculation the 5th week of chicken, two kinds of vaccine virus strains all in conjunctiva, copy but
it is positive in UNI L group, only having a chicken.
vITAPEST L vaccine virus but non-
uNI L similarly copies in other organs.
Similarly, the applicant has proved vaccine virus strain retaining in conventional laying hen.Especially, be presented at after eye inoculation first week of chicken,
the higher level copying in UNI L group.In the time that chitosan is used as adjuvant, in more organs and within the longer time, be separated to virus.At after eye inoculation the 5th week of chicken, only in the conjunctiva of some vaccinated conventional laying hens, be separated to two kinds of vaccine virus strains (Fig. 5).
Finally, the applicant proves in the following Example 4, with common observe in mammal contrary, in poultry, be not enhanced by the vaccine-induced humoral immunization of difference, thereby when by the time that eye approach is used to birds, show the outstanding especially effect of the vaccine combination with chitosan.
Illustrate (but restriction never in any form) the present invention by the following example.
Embodiment
Embodiment 1: materials and methods
Embodiment 1.1: chicken
Respectively from by Lohmann Valo (Cuxhaven, Germany) and the ovum that provides of Wijverkens hatchery (Halle, Belgium) hatch SPF white leghorn and conventional laying hen (SSL:sex sale linked).Obtain the ovum of conventional laying hen from the breeder flock (breeder flock) in 28 week age, described breeder flock is accepted following ND vaccination regimen: in the time of 4 and 8 week age, inoculated with NOBILIS Clone30 (Intervet), then in the time of 16 week age, strengthened with NOBILIS Newcavac (vaccine of deactivation, Intervet).After hatching, whole chickens are remained in biosafety level 3 (BSL-3) isolator, under the mandate of the Biosafety and Bioethics Committees of Veterinary and Agrochemical Research Institute and supervision, carry out zoopery according to country and European detailed rules and regulations.
Embodiment 1.2: vaccine, adjuvant and attack strain
Provided by CEVA-PHYLAXIA Veterinary Biologicals Co Ltd (Hungary)
vITAPEST L attenuated vaccine and chitosan (chitosan hydrochlorate) adjuvant.Vaccine is based on asymptomatic PHY.LMV.42 strain (Meszaros J.Aerosol-vaccination against Newcastle disease.Deut Tierarztl Woch1991; 98:117-164; Meszaros J, Szemeredi M, Tamasi G.Immunization of day-old chickens against Newcastle disease.Acta Vet Hung1992; 40:121-127); it belongs to genotype I (Czegledi A; Ujvari D; Somogyi E; Wehmann E; Werner O, Lomniczi B.Third genome size category of avian paramyxovirus serotype1 (Newcastle disease virus) and evolutionary implications.Virus Res2006; 120:36-48).In phosphate buffered saline(PBS) (PBS buffer), reconstruct vaccine batch to obtain 1 dosage in 50 μ l, and it is corresponding to ≈ 10
6eID
50/ dosage.The chloride of the not branch binary heteropolysaccharide that chitosan hydrochlorate is made up of N-acetyl-GLUCOSAMINE and the GLUCOSAMINE of Liang Ge unit.It is by conventionally causing the chitinous partially deacetylated acquisition of 70% to 95% degree of deacetylation.Final concentration with 0.5% (w/v) is dissolved in chitosan in PBS buffer, uses it for reconstruct and dilution vaccine to final concentration.At Mexico (Calderon NL; Galindo-Muniz F; Ortiz M; Lomniczi B; Fehervari T, Paasch LH.Thrombocytopenia in Newcastle Disease:haematological evaluation and histological study of bone marrow.Acta Vet Hung2005; 53 (4): 507-513) separate the Chimalhuacan NDV strain for attacking, it belongs to II genoid type V.It is the anaphylactic type viscerotropism strain with 1.89 ICPI.10
5eID
50this strain eye nose or be directly in 3 to 6 days, to induce in the SPF chicken attacked in 3 to 6 weeks age and business maternal immunity broiler 100% mortality rate (individual's observation) through eye inoculation.
Embodiment 1.3: mitogen and NDV antigen
Mitogen phorbol 12-myristate 13-acetas (PMA) and ionomycin (Iono) are purchased from Sigma (Belgium).(Lambrecht B as described above; Gonze M; Meulemans G, van den Berg T.Assessment of the cell-mediated immune response in chickens by detection of chicken interferon-gamma in response to mitogen and recall Newcastle disease viral antigen stimulation.Avian Path2004; 33:343-350) prepare NDV recall antigen from NDV La Sota strain, called after NDV glycoprotein (gp-NDV).
Embodiment 1.4: the tissue distribution of vaccine strain and the measurement copying
From conjunctiva (it shows more susceptible of NDV) (the Nakamura K of palpebra inferior; Ohta Y; Abe Y; Imai K, Yamada M.Pathogenesis of conjunctivitis caused by Newcastle disease viruses in specific-pathogen-free chickens.Avian Path2004; 33 (3): 371-376), trachea, lung, spleen, pancreas, duodenum and collection organization of caecal tonsils sample, and kept freezing until process.
the tissue distribution of VITAPEST L vaccine strain and copying by being specific to
the QRRT-PCR of VITAPEST L measures, and described QRRT-PCR allows to detect 10
2eID
50/ reaction (10
4.18eID
50/ 20mg tissue).Result is expressed as the vaccine strain titre of every 20mg tissue.As the people such as Van Borm S, A universal avian endogenous real-time reverse transcriptase-polymerase chain reaction control and its application to avian influenza diagnosis and quantification.Avian Dis2007; 51:213-220 is described, makes birds α-actin confirm the quality of RNA extracting method and sample as housekeeping gene.
Embodiment 1.5: the immune measurement of immunocompetence and the mediation of NDV specific cell
Respectively by the mitosis of splenocyte and the immunity of NDV antigen activation investigation immunocompetence and the mediation of NDV specific cell.In brief, sterilely from chicken, take out spleen, separating Morr. cell, positive control with mitogen (0.1 μ g/ml PMA/Iono) activation splenocyte as the in vitro activability of splenocyte, or activate splenocyte with NDV recall antigen (gp-NDV) (1 μ g/ml).Cell response is expressed as optical density (O.D.) and is worth and is equal to or greater than 0.1 O.D and be considered to the significantly evidence of activation.
Embodiment 1.6:NDV specificity humoral and local antibody-mediated immune measurement
Suppress (HI) test and estimate NDV specific humoral immunity by NDV specific IgG, IgA and IgMELISA by hemagglutination.Measure the immunity to NDV of local antibody's mediation in tear, lung, bile and duodenum by ELISA.
Embodiment 1.7: the measurement of the oropharynx of aggressivity NDV strain and cloaca excretion
Oropharynx and cloaca cotton swab are immersed in the 1ml brain heart perfusion sampling buffer (37g is dissolved in 1 liter of distilled water) (Becton Dickinson Benelux, Belgium) that is supplemented with antibiotic (10000IU/ml penicillin, 2mg/ml streptomycin, 1mg/ml gentamycin and 650 μ g/ml kanamycin).Cotton swab is preserved at-80 DEG C until further analyze.Analyze in order to utilize quantitatively reverse transcription-polymerase chain reaction (QRRT-PCR) in real time, use MagMAX96AI/ND viral RNA Ambion test kit (Applied Biosystems according to the scheme of manufacturer, Lennik, Belgium) extract RNA at the upper cotton swab from 50 μ l submergences of KingFisher magnetic-particle processor (Thermo Scientific).The RNA of purification is eluted in 50 μ l elution buffers.
By thering is the quantity that quantitatively reverse transcription-polymerase chain reaction (QRRT-PCR) mensuration Chimalhuacan NDV attacks the substrate gene copy of strain in real time as above of little change.In brief, activate after step in initial reverse transcription step and thermal starting Taq-, on Applied Biosystems7500 PCR in real time circulating instrument, carry out 50 circulations (carry out 15 seconds, carry out 34 seconds, carry out 10 seconds) at 72 DEG C at 54 DEG C at 95 DEG C.Primer (M+4100 and M-4220) and MGB-Taqman probe (M+4169FAM-TAMRA) act on substrate gene and synthetic by Eurogentec (Liege, Belgium).By contrasting with the standard curve being formed by the total viral rna of Chimalhuacan NDV strain the virus titer of measuring each sample.Take turns experiment at each and comprise this standard curve.Based on the result of standard curve, be specific to the threshold of sensitivity (R of the NDV QRRT-PCR of Chimalhuacan NDV strain
2=0.998, efficiency=94.17%) be measured as 10
1eID
50/ reaction.It means QRRT-PCR and reacts completely and can be comparable to higher than 10
2.7eID
50/ ml wipes sample and Quantitative Comparison is feasible.Result is expressed as every ml wipes the titre of the attack strain of sample.
In addition; as the people such as Van Borm S, A universal avian endogenous real-time reverse transcriptase-polymerase chain reaction control and its application to avian influenza diagnosis and quantification.Avian Dis2007; Described in 51:213-220, make birds α-actin verify the quality of sample and RNA extracting method.
Embodiment 1.8: experimental design
SPF chicken is carried out to two similar experiments (experiment I and II).In the time of 1 age in days, 105 SPF chickens are divided into 3 groups in BSL-3 isolator, 35 every group.On the same day, use respectively the use of single dosage or do not use 0.5% chitosan as adjuvant
vITAPEST L vaccine is by two groups of eye nose approach inoculation.By directly using and carry out similar experiment (data do not show) through eye.The 3rd group keeps not inoculation and is used as negative control group.Inoculation rear (p.v.) 2 days, people is genuine kills chicken to obtain blood, bile and duodenum.From p.v.1 to 5 week, collect tear with the interval of a week, and gather blood, spleen, bile and duodenum sample from identical animal.At the 1st and the 5th week, in an experiment, additionally obtain conjunctiva, trachea, lung, pancreas and caecal tonsils.At the 3rd and the 5th week, painlessly collecting lung cleaning materials from the other chicken of each group after lethal by rather must appropriate (CEVA Sant é Animale) injecting.On each time point, use 5 chicken/groups.In experiment II, within the 2nd, 3,4 and 5 weeks, obtain tear, blood, spleen, bile and duodenal sample at p.v..
Also in conventional laying hen, carry out 2 other experiments (experiment III and IV).In the time of 1 age in days, 90 conventional laying hens are dispensed to 3 groups in BSL-3 isolator, each organizes 30 chickens.On the same day, inoculate as described above two groups; The 3rd group keeps not inoculation and is used as negative control group.Except only within the 5th week, collecting lung cleaning materials at p.v., the experiment install and experiment I of experiment III is similar.In experiment IV, within the 2nd, 3,4 and 5 weeks, obtain tear, blood, spleen, bile and duodenal sample at p.v..In the time of 5 week age, by eye nose approach with 10
5eID
5010 chickens of each group are attacked in the Chimalhuacan NDV strain of/200 μ l.One by one identify chicken.After attack, monitor chicken with regard to clinical symptoms (edema of head, dispirited, collapse and neurosigns) and mortality rate every day.After attack, (p.ch.) obtains oropharynx in the 2nd, 4,7 and 10 days and cloaca is wiped sample.P.ch. the 11st day, the chicken of painless lethal survival and collect blood sample.Obtain spleen and duodenum from 5 chicken/groups.
Embodiment 1.9: statistical analysis
Use as described above Minitab13 and STATA10 software (for the statistical procedure of Windows2000) to carry out statistical analysis, and difference is considered to significant in the time of P<0.05.
Embodiment 2: the tissue distribution of vaccine strain and copying
All for α-actin, endogenous amplification contrast shows positive amplified signal (Ct<40) to organ samples, thereby has confirmed their fine quality and RNA purity.
In whole organ samples p.v. the 1st week vaccinated SPF chicken, detect
vITAPEST L virus (table 1).P.v. the 5th week, only in conjunctiva, lung, spleen, duodenum, pancreas and the caecal tonsils of some chickens, vaccine strain detected.In the time using chitosan, vaccine strain detected more continually; But, nothing difference statistically between virus titer.Inoculation 1 age in days conventional laying hen after 1 week,
vITAPEST L vaccine strain is present in conjunctiva, trachea and duodenal several sample (table 1).In the time that vaccine uses chitosan as adjuvant, also in some lungs, spleen and caecal tonsils's sample, vaccine strain detected.However,, in vaccinated conventional laying hen, detect frequency and titre lower than what find in the SPF chicken of inoculation.P.v. the 5th week, copying of vaccine virus in conjunctiva, still can be detected.
table 1: with thering is or do not have chitosan adjuvant
the detection (experiment I and III respectively) of the vaccine virus after VITAPEST L vaccination 1 age in days SPF chicken and conventional laying hen.
ap.v. specify time on by QRRT-PCR to 20mg tissue test data.For this tissue, there is significantly (P<0.05 or 0.017 uses Bonferroni to proofread and correct) of different upper target data differences.Be specific to
the cutoff value of the QRRT-PCR of VITAPEST L vaccine strain is measured as 10
4.18/ 20mg tissue.
bfrequency that in data representation 20mg tissue, virus detects (positive quantity/always test quantity).
cthe log of NDV in the tissue of the positive chicken of data representation 20mg
10eID
50mean+SD.
Embodiment 3: the immunity that immunocompetence and antigenic specificity are cell-mediated
The result about the splenocyte activation that uses PMA/Iono mitogen obtaining on different sampling time points shows, in each group, all SPF chicken has similar immunocompetence (O.D.>0.1) with conventional laying hen in each experiment of 4 experiments, thereby has verified the activability of splenocyte.In fact, chitosan does not affect cellullar immunologic response, as by not having any significant difference between 2 vaccinated group in the experiment I for SPF chicken and conventional laying hen (Fig. 6 A) and IV (Fig. 6 B) respectively and being proved.In the time that work ND vaccine and chitosan are used altogether, this unaltered immunocompetence is verified (data do not show) in experiment II and III.
In SPF chicken (experiment I),
vITAPEST L strain can induction NDV specific cell mediation in p.v. the 1st week immune measurable activation and from after second week vaccinated group with nonvaccinated matched group significant difference (P<0.05) (Fig. 6 A).In the time supplementing chitosan, use
the eye nose immunity of VITAPEST L vaccine produces remarkable higher antigenic specificity cellular immunization for thoughtful the 4th week from p.v. the 1st and promises (P<0.05), as the ChIFN γ output of the increase by splenocyte is proved.In SPF chicken, chitosan is confirmed (data do not show) to the immune positive effect of specific cell mediation in experiment II.
In conventional laying hen (experiment IV), within the 3rd week, start all to observe from p.v. the immunity (P<0.05) (Fig. 6 B) that NDV specific cell mediates the group of two inoculations.In the time that chitosan and ND live vaccine are used altogether, with independent inoculation
vITAPEST L vaccine is compared, and this NDV specific cell is replied higher, although not remarkable.In experiment III, obtain identical result (data do not show).
Embodiment 4: body fluid and local antibody-mediated immunity
Within the 1st week, start by specific IgM ELISA from p.v., but within the 2nd week, start by hemagglutination inhibition test and the induction (table 2) of recording humoral immunization vaccinated SPF chicken by IgG and IgA specific ELISA from p.v., titre is different from nonvaccinated chicken statistically.The NDV specificity HI obtaining in conventional laying hen and the feature of IgG antibody titer are corresponding to the decline of passive maternal immunity, until 4 week age, and within the 5th week, start the primary immune response (table 2) that can detect that progressive active is vaccine-induced from p.v..In the serum of conventional animal, fail to detect NDV specificity IgA and IgM (data do not show).Chitosan does not all show the adjuvant effect to humoral immunoresponse(HI) in SPF chicken and conventional laying hen, as what proved by any statistically significant difference that does not have titre between two vaccinated group.
table 2: with thering is or do not have chitosan adjuvant
humoral immunization (experiment I and III respectively) after SPF chicken and the conventional laying hen of VITAPEST L vaccination 1 age in days.
Data be illustrated in p.v. specify time on respectively by the absorbance of HI and ELISA measurements determination and the log reciprocal of serum dilution
2mean+SD (n=5).With 1:100 by serum samples diluted.There is the mean+SD significant difference (P<0.05) on different upper target time points.
Chitosan is alterable height to the antibody-mediated immune effect of tear of SPF chicken (Fig. 7 A) and conventional laying hen (Fig. 7 B), and with thering is or do not have (w/o) chitosan
difference between the chicken of VITAPEST L inoculation is not statistically significant on each time point., bile and duodenal antibody-mediated immunity relevant for the lung of SPF chicken, observe the similar variation (table 3, respectively, Fig. 8 A and 8A) of the effect of chitosan adjuvant.In conventional laying hen, in bile the 1st week (Fig. 9 B) and in duodenum from 1 week age until source of parents NDV specific IgG antibodies detected 5 week age (Fig. 9 B).P.v. the 5th week, duodenum IgG replied in vaccinated group significantly higher, thereby shows the primary immune response that active progressive in digestive tract is vaccine-induced.Chitosan does not have active effects with the antibody-mediated immunity to duodenum of using altogether of the ND vaccine of living.
Table 3: with thering is or do not have chitosan adjuvant
the antibody-mediated immunity (experiment I) of lung after VITAPEST L vaccination 1 age in days SPF chicken.
Data are illustrated in the mean+SD (n=5) of the upper absorbance of measuring by ELISA of time of p.v. appointment.Respectively, for IgA/G and the undiluted lung cleaning materials of IgM sample.There is the mean+SD significant difference (P<0.05) on different upper target time points.
In the experiment II repeating and IV, in SPF chicken and conventional laying hen, all confirm this unaltered general and the local antibody response (data do not show) for the ND vaccine alive of using altogether with chitosan.
Embodiment 5: the protective effect after attack and draining again
The chicken of all being attacked in nonvaccinated group all shows clinical sign, and it starts from attacks rear the 3rd day (d.p.ch.) and death between 4 to 6d.p.ch.
in the group of VITAPEST L inoculation, two animals show that clinical sign and one are in 7d.p.ch death.With using altogether with chitosan
the chicken of VITAPEST L inoculation does not show clear and definite M & M in experimentation.
The discharge (shedding) of challenge virus starts from 2d.p.ch. and all oropharynxs and cloaca in matched group, and to wipe sample be positive (table 4) at 4d.p.ch.On each time point, the discharge of being undertaken by oropharynx and cloaca approach reduces because of inoculation.In addition the minimizing of comparing of the group of, accepting the quantity of the birds draining in the group of the ND live vaccine used altogether with the chitosan vaccine independent with acceptance.Excretion almost completes and stops completely at 10d.p.ch. at 7d.p.ch..
table 4: attacking with thering is or do not have chitosan adjuvant
the discharge of Chimalhuacan NDV strain after the conventional laying hen of VITAPEST L vaccination (experiment IV).
aby the sample of wiping gathering on the time of specifying at p.ch. of 1ml is carried out QRRT-PCR and carrys out determination data.There are different upper target data and wipe sample significant difference (P<0.05 or 0.017 uses Bonferroni to proofread and correct) for this.The cutoff value that is specific to the QRRT-PCR of Chimalhuacan NDV strain is determined as 10
2.7/ ml wipes sample.Due to specific mortality rate, the total quantity of the chicken of test passs in time and reduces (about detailed content, referring to result part).N.D.=is undeterminate due to specific mortality rate.
bfrequency that the virus of wiping data representation 1ml in sample detects (positive quantity/always test quantity).
The application relates to following embodiment:
1. be suitable for using the vaccine combination with the described avian species of immunity to avian species through eye the chitosan that it comprises one or more vaccines and effective adjuvant amount.
2. for the vaccine combination that comprises one or more vaccines and the chitosan of effective adjuvant amount of the described avian species of immunity, it is used by eye drop, spray or aerosol.
3. the vaccine of project 1 or 2, wherein separately or use chitosan and the vaccine of effective dose simultaneously.
4. the vaccine of any one of aforementioned project, wherein one after the other uses chitosan and vaccine to avian species by eye drop, spray or aerosol, and chitosan was used before or after vaccine.
5. the vaccine combination of any one of aforementioned project, wherein said vaccine is virus or antibacterial or parasite.
6. the vaccine combination of any one of project 1 to 5, wherein said vaccine is attenuated microorganisms, killed deactivation microorganism, the recombinant microorganism of living, microorganism, its subunit or its combination of complete attenuation or deactivation or restructuring.
7. the vaccine combination of any one of aforementioned project, wherein said vaccine derives from and causes common poultry disease as the virus of fowl pox, newcastle, infectious bronchitis, lymphatic system leucocyte hyperplasia, Marek, infectious bursal disease, infectious laryngotracheitis, Reovirosis, infectious tenosynovitis, birds encephalomyelitis, swollen a syndrome, turkey rhinotracheitis or bird flu, or derives from and cause the antibacterial of mycoplasma, hemorrhagic septicemia, salmonellosis, Bordetellosis and/or derive from the parasite that causes coccidiosis, campylobacteriosis.
8. the vaccine combination of any one of project 1 to 7, wherein said avian species is poultry or the bird of raising or raise and train, and comprises chicken, turkey, goose, duck, pheasant, Carnis Coturnicis japonicae or pigeon.
9. the vaccine combination of any one of project 1 to 8, it is retaining in birds tissue for increasing immunity cell-mediated in avian species and vaccine.
10. chitosan and vaccine are for the preparation of the purposes of vaccine combination, described vaccine combination is replied with vaccine and is organized retaining in pathogen birds for increasing cell-mediated bird immunity, wherein uses described vaccine combination by eye drop, spray or aerosol to birds.
The purposes of 11. projects 10, wherein uses chitosan and vaccine to avian species dividually or side by side.
The purposes of 12. projects 11, wherein passes through eye drop, spray or aerosol to avian species sequential application chitosan and pathogen, and chitosan was used before or after vaccine.
The purposes of any one of 13. projects 10 to 12, wherein said vaccine is birds parasite, virus or antibacterial.
The purposes of any one of 14. projects 10 to 13, wherein said vaccine is attenuated microorganisms, the killed deactivation microorganism of living, complete attenuation or the microorganism of deactivation, its subunit or its combination.
The method of 15. immune avian species, it comprises the step that the vaccine combination of any one of project 1 to 9 is applied to the ophthalmic of avian species.
16. increase cell-mediated immunity and the method that retain of vaccine in birds tissue in avian species, and it comprises the step that the chitosan of one or more vaccines and effective adjuvant amount is applied to the ophthalmic of avian species.
The method of 17. projects 15 or 16, wherein dividually, one after the other or side by side uses vaccine and chitosan.
The method of any one of 18. projects 15 to 17, is wherein used and is carried out using through eye to birds by spraying, aerosol or eye drop.
The method of any one of 19. projects 15 to 18, wherein after maternal antibody (MDA) weakens by be administered to the reinforcement that birds inoculates through eye.
The method of any one of 20. projects 15 to 18, wherein after birds inoculation for the first time or with it simultaneously by the birds in 1 age in age in days to 4 week is carried out to the reinforcement of using to inoculate through eye.
21. 1 kinds of birds vaccinating agents boxes, it comprises the distributor that the eye of the vaccine combination of any one that is suitable for project 1 to 9 is sent with eye drop, spray or aerosol.
The birds vaccinating agents box of 22. projects 21, wherein said distributor is aerosol, spray or eye drop.
The birds vaccinating agents box of 23. projects 21 or 22, it comprises the chitosan of effective dose and the container separating of vaccine is housed respectively.
The birds vaccinating agents box of any one of 24. projects 21 to 23, it also comprises having using and the technical instruction of the information of dosage about vaccine combination.
Claims (7)
1. comprise the compositions of attenuation Avian pneumo-encephalitis virus alive and chitosan for the preparation of the purposes of vaccine combination, described vaccine combination is for being administered to birds by eye drop, spray or aerosol through eye, thus immunity or inoculate described birds.
2. the purposes of claim 1, wherein separately or use the attenuation Avian pneumo-encephalitis virus of described chitosan and described work simultaneously.
3. the purposes of claim 1 or 2, wherein one after the other uses the attenuation Avian pneumo-encephalitis virus of described chitosan and described work to avian species by eye drop, spray or aerosol, described chitosan was used before or after the attenuation Avian pneumo-encephalitis virus of described work.
4. the purposes of claim 1, wherein said vaccine combination is selected from following avian species for immunity or inoculation: poultry raising or that raise and train or bird, comprise chicken, turkey, goose, duck, pheasant, Carnis Coturnicis japonicae or pigeon.
5. the purposes of claim 1, wherein said vaccine combination is retaining in birds tissue for increasing immunity cell-mediated in avian species and described virus.
6. chitosan and the attenuation Avian pneumo-encephalitis virus of living be for the preparation of the purposes of vaccine combination, and described vaccine combination is by via the using through eye of eye drop, spray or aerosol, and immunity or inoculation birds.
7. the purposes of claim 6, wherein dividually or side by side use the attenuation Avian pneumo-encephalitis virus of chitosan and described work to birds.
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| CN102973936A (en) * | 2011-12-14 | 2013-03-20 | 普莱柯生物工程股份有限公司 | Application of chitosan in avian vaccine composition preparation |
| EP2644702A1 (en) | 2012-03-30 | 2013-10-02 | Ceva Sante Animale | Multivalent recombinant avian herpes virus and vaccine for immunizing avian species |
| CN102824635A (en) * | 2012-08-20 | 2012-12-19 | 上海生物制品研究所有限责任公司 | Application of chitosan as adjuvant in preparation of influenza virus attenuated live vaccine |
| ES2936811T3 (en) | 2013-03-12 | 2023-03-22 | Wellstat Vaccines Llc | Conjugate to induce antibodies directed to fungal cell wall polysaccharides |
| WO2014174039A1 (en) * | 2013-04-24 | 2014-10-30 | Medizinische Universität Wien | Novel vaccine formulation for ocular immunization |
| WO2015116502A1 (en) * | 2014-01-31 | 2015-08-06 | The Regents Of The University Of California | Detection and treatment of gnaq mutant uveal melanoma cells with gold nanoparticles |
| RU2583886C2 (en) * | 2014-02-28 | 2016-05-10 | Пётр Евгеньевич Игнатов | Multipotent vaccine for prevention and treatment of primarily invasive and infectious diseases, method of use |
| CN104857013A (en) * | 2015-03-31 | 2015-08-26 | 中国科学院海洋研究所 | Drug capable of inhibiting Newcastle disease virus |
| CN107446858B (en) * | 2017-09-02 | 2020-11-03 | 河南省农业科学院畜牧兽医研究所 | Pigeon mycoplasma and application thereof |
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| DE602004020189D1 (en) * | 2003-01-30 | 2009-05-07 | Novartis Vaccines & Diagnostic | ADJUVANT INFLUENZA VACCINE |
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| US20110223195A1 (en) | 2011-09-15 |
| PL2331128T3 (en) | 2014-06-30 |
| BRPI0918806A2 (en) | 2016-11-01 |
| BRPI0918806B1 (en) | 2019-02-05 |
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